It is presently known in the worldwide industry the need to produce biodegradable and biocompatible materials by using renewable raw materials and energy sources through processes that are not aggressive to the environment.
In the modern society, although the use of plastic materials in a large scale has represented a mark in the history of technological development, the increasing utilization of these materials is leading to a diversity of serious environmental problems. In the case of the industry of petrochemical-derived plastic resins, the annual amounts produced are of about 200 millions tons. These materials, which are very resistant to natural degradation, rapidly accumulate in the disposal areas mainly around the large urban centers. In view of these problems, the development of biodegradable plastic resins has received worldwide attention, mainly those produced by means of a clean technology using renewable sources. Considering the relevance of these facts, the market potential for using these new materials is enormous. The applications of these biodegradable biopolymers with greater chances of success in the market involve products, such as disposable materials, for example packages, cosmetic and toxic agrochemical recipients, medical and pharmaceutical articles, etc.
An important family of the biodegradable biopolymers is the Polyhydroxyalkanoates (PHAs), which are polyesters naturally synthesized by a large number of live beings. With more than 170 representatives described in the literature, the commercial interest in the PHAs is directly related not only to the biodegradability but also to their thermo-mechanical properties and production costs. Thus, only some PHAs have found industrial application, most representatives being the PHB (poly-3-hydroxybutyrate), PHB-V (poly(hydroxybutyrate-co-hydroxyvalerate)), P4HB (poly(4-hydroxybutyrate)), P3HB4HB (poly(3-hydroxybutyrate-co-4-hydroxybutyrate)) and some PHAmcl (polyhydroxyalkanoates of medium chain), the typical representative of this last family being PHHx (polyhydroxyhexanoate).
The chemical structure of the PHAs may be described as a polymeric chain formed by repetitions of the following unit:
Where R is an alkyl or alkenyl group of variable length and m and n are integers, in the polymers mentioned above R and m assuming the following values:PHB: R=CH3, m=1PHB-V: R=CH3 or CH3-CH2-, m=1P4HB: R=H, m=2P3HB-4HB: R=H or CH3, m=1 or 2PHHx: R=CH3-CH2-CH2-, m=1
Most PHAs may be processed in conventional extrusion and injection equipments, without requiring significant modifications for a good processing. It is also possible to process these polymers in cast and coating film systems to be used as packaging materials for the food industry, for example.
As a function of the development stage of these polymers, it is possible to use them to produce packages for personal hygiene products of short use and with low grammage. They can also be used to manufacture containers and packages for agrochemicals, engine oils, disposable diapers, and the like. Moreover, where the intrinsic property of biodegradability is required, the PHAs are applicable according to well defined technical and commercial aspects, such as: garbage bags, golf tees, fishing articles and other products directly connected to the handling of plastic materials in open air.
In agro-industry, the PHAs may be applied to plant pots, reforestation tubes, greenhouse and coverage films and mainly to controlled release system nutrients, fertilizers, herbicides and insecticides.
For biomedical applications, the PHAs can be used for microencapsulating drugs of controlled release, medical sutures and fixation pins for bone fractures, due to their total biocompatibility and to the small reaction from the receiving organism to the presence of a strange body. Furthermore, with a in vivo biodegradation rate which is very slow but continuous and complete, the PHAs present an excellent potential to be applied as a basic structure for re-absorbable prostheses.
The great development of the natural sciences in the last two decades, particularly in biotechnology, has allowed the use of most different natural or genetically modified organisms in the commercial production of PHAs. Particularly relevant for the present invention is the use of determined bacterial strains which are able to produce and to accumulate expressive quantities of these polymers in their interior. Cultivated in specific conditions, which allows reaching high cellular density, high content of intracellular polymer, and yields compatible with the industrial process, these bacterial strains can use different renewable raw materials, such as sugarcane, molasses or hydrolyzed cellulose extracts.
Although attempts have been made for applying the bacterial cells in natura (without using PHA solubilizing agents) as moldable material, such as disclosed in U.S. Pat. No. 3,107,172, the commercial applications of PHAs in most cases require a sufficiently high purity to attain the desired plastic properties. In order to achieve the adequate levels of purity for processing the biopolymer, specially the PHAs, there are normally required steps in which the utilization of solvents for extraction and recovery of the PHA from the residual biomass is indispensable.
In patent EPA-01455233 A2, there are described several possibilities to carry out the digestion of an aqueous suspension of cells containing PHA, using enzymes and/or surfactants to solubilize the non-PHA cellular material. This patent mentions as a possible restriction to the processes that use solvent the fact that they require large quantities of solvents and therefore have high production costs. Nevertheless, it mentions that the solvent step is not eliminated, if a product of high purity is desired. Furthermore, although the enzymes used in this process are added in relative low quantities (1% in relation to the dry cell material) they are very expensive and cannot be recovered in the process, contrarily to what occurs when a solvent is used. Also, high dilution of the cellular material is required, which leads to a high volume of effluents generated in the process.
The usually proposed extraction processes basically consist in exposing the dry or humid cellular biomass containing the biopolymer in a vigorous contact with a solvent that solubilizes it, followed by a step where the cellular residue is separated. The solution containing the biopolymer then receives the addition of an insolubilizing agent, which induces its precipitation in the solvent (see, for example, Brazilian patent PI 9103116-8 filed on Jul. 16, 1991 and published on Feb. 24, 1993.
In the extraction processes through organic solvents often cited in the literature for extraction and recovery of PHA from bacterial biomass, the solvents utilized are partially halogenated hydrocarbons, such as chloroform (U.S. Pat. No. 3,275,610), methylene-ethanol chloride (U.S. Pat. No. 3,044,942), chloroethanes and chloropropanes with boiling point within the range from 65 to 170° C., 1,2-dichloroethane and 1,2,3-trichloropropane (patents EP-0014490 B1 and EP 2446859).
Other halogenated compounds, such as dichloromethane, dichloroethane and dichloropropane are cited in U.S. Pat. No. 4,562,245 (1985), U.S. Pat. No. 4,310,684 (1982), U.S. Pat. No. 4,705,604 (1987) and in European patent 036.699 (1981) and German patent 239.609 (1986).
The processes of extraction and purification of biopolymers from biomass which utilize halogenated solvents are totally prohibitive nowadays, since they are highly aggressive to the environment and to human health. Therefore, a solvent to be used as a potential extractor of the biopolymer from a cellular biomass should first fulfill the condition of not being aggressive to the environment.
In this sense, Brazilian patent PI 9302312-0 (filed on 1993 and granted on Apr. 30, 2002) presents a process of extracting biopolymer from bacterial biomass which employs as solvents high chain alcohols with 3 carbons or the acetates derived therefrom. This patent prefers isoamyl alcohol (3-methyl-1-butanol), amyl acetate (or amyl-acetic ester) and fusel oil, a mixture of high alcohols obtained as a by product of the alcoholic fermentation and which has as main component the isoamyl alcohol. This patent is also characterized for using a single solvent as extractor and purifier, not requiring the utilization of an insolubilizing agent or counter-solvent and/or marginal non-solvent. The precipitation of the solute (biopolymer) of the PHA solution is carried out through the cooling of the solution.
The U.S. Pat. No. 6,043,063 (filed on Apr. 14, 1998 and granted on Mar. 28, 2000), U.S. Pat. No. 6,087,471 (filed on Apr. 14, 1998 and granted on Jun. 11, 2000) and the international patent application WO-98/46783 (filed on Apr. 15, 1997) discloses an extensive list of non-halogenated solvents which could be potentially used as solvents for extracting biopolymer from biomass, but many of them presenting characteristics such as difficult industrial manipulation, toxicity, besides high cost. In said extensive list, which also includes the solvents cited in Brazilian patent PI 9302312-0, only a small number of solvents have potential to be industrially used for extracting biopolymer from vegetal or bacterial biomass, either due to problems regarding incompatibility with the biopolymer, or due to their toxicity, explosiveness, and also high cost. Moreover, Brazilian patent PI 96102256, filed in Brazil on Aug. 16, 1996 and published on Jul. 6, 1999 is even more selective, since it includes compounds that are highly noxious to human health, besides mineral and vegetal oils, carbonic gas (of super critical and expensive extraction technology) among others, as probable solvents useful to extract biopolymer from vegetal or bacterial biomass. At the same time, this patent contemplates the necessity of avoiding solvents that are potentially harmful to health and to the environment.
Since the biopolymers are heat sensitive, i.e. when submitted to temperatures above a determined value, they degrade irreversibly, losing molecular weight, which can definitely affect the properties that characterize them as thermoplastics, it is fundamental to have in mind that the list of solvents with potential to be industrially used becomes even more restrict.
The potential for industrial utilization of the solvent selected to promote the extraction of the biopolymer will be increased if it is associated with an adequate process that allows extracting the biopolymer without causing significant alterations in its molecular weight. Remarkably, in the case of the solvent which needs to be heated above 70° C. to solubilize the biopolymer, the longer it remains exposed to this temperature during the processing, the more it will degrade, which fact can irremediably impair its thermoplastic properties. The lesser alteration the PHA suffers during the process of extraction, the wider will be the range of its possible commercial applications.
As taught in the literature, the kinetics of degradation of the biopolymer, especially the PHA, obeys to a zero-order reaction (see for example the master's degree thesis: Berger, E., ‘Elaboration des techniques de separation pour des biopolymeres d'origine bacterienne: les acides poly-β-hydroxyalcanoiques’, Departement de Genie Chimique-Ecole Polytechnique—Universite de Montreal, Canada, 1990, pages 72-75). Considering the ratio of degradation of its molecular weight to the time it is exposed at a temperature T as dMW/dt, the equation that defines this degradation is:(dMW/dt)T=k  (1)where:k: is a constant for a given solvent at a given temperature T.thus, if the equation (1) is integrated for a time interval 0-t, we have:MWT=k·t+MWo  (2)Where:MWT: is the molecular weight of the biopolymer after the time of extraction t, for a given temperature T, has elapsed, in a solvent S;MWo: is the molecular weight of the biopolymer contained in the biomass, at the time t=0, before being submitted to the extraction;K: is a constant of proportionality for a given temperature T and solvent S.
By way of example, 20 g biomass of dry Alcaligenes eutrophus, containing 70% PHB on a dry base are mixed with 1500 g of isoamyl alcohol (technical grade) at 110° C., submitting the mixture to different times of extraction and filtration for removing insoluble particles from the biomass. The obtained PHB solution is then rapidly cooled to 30° C. to guarantee the precipitation of the PHB, which is subsequently filtrated and dried in air stream at room temperature until the complete depletion of the solvent. Then, the PHB is submitted to molecular weight evaluation by the GPC technique (Gel Permeation Chromatography) to result, after a mathematic adjustment through linear regression, in the following equation of degradation:MWT=−9753.81·t+1,000,000, R2=0.98  (3)Where:MWT: is the molecular weight of the polyhydroxybutyrate after the extraction in isoamyl alcohol at 110° C., in Daltons;T: is the time, in minutes, of exposure of the polyhydroxybutyrate to a temperature of extraction of 110° C. in isoamyl alcohol;R: is the coefficient of correlation of the experimental points with the equation of adjustment.
Thus, from equation (3) we have that the polyhydroxybutyrate, originally containing a molecular weight of 1,000,000 Da and submitted to an extraction in isoamyl alcohol at 110° C. would give, for a time of 5 minutes, a molecular weight of 951,230 Da; for 15 minutes of exposure, 853,692 Da; for 30 minutes of exposure, 707,410 Da; for 60 minutes, 414,771 Da; and for 90 minutes, 122,230 Da.
Considering that besides the extraction other operations such as evaporation and drying of the solvent are necessary to obtain a pure product with good mechanical properties, and that these operations many times expose the biopolymer to critical situations regarding the material, it is not difficult to imagine the inherent difficulties of processing this type of material. Besides the solvent, it is desirable to have an adequate process which does not degrade the product thermally.
Thus, for purposes of exemplification, the solvents mentioned in U.S. Pat. No. 6,043,063 and their respective temperatures of PHA extraction, at Celsius degrees between parenthesis, are presented in the list below: ethyl butyrate (120° C.), propyl propionate (118° C.), butyl acetate (120° C.), butyl propionate (123° C.), tetrahydrofurfuryl acetate (121° C.), methyl propionate (75° C.), normal-methyl valerate (115° C.), 1-butanol (116° C.), 2-methyl-1-butanol (117° C.), 3-methyl-1-butanol (125° C. and 126° C.), 1-pentanol (125° C. and 126° C.), 3-pentanol (115° C.), amyl alcohol (128° C.), 1-hexanol (134° C.), ethylene glycol diacetate (137° C.), tetrahydrofurfuryl alcohol (117° C.), methyl-amyl-ketone (120° C.), methyl-isobutyl-ketone (115° C.), acetophenone (110° C.), 1,2-diaminopropane (115° C.), alpha-methylstyrene (126° C.), dimethyl sulfoxide (117° C.), propylene carbonate (110° C.), 1,2,3-trimethyl-benzene (121° C.), dimethyl acetamine (90° C.) and dimethylformamide (90° C.). These solvents will have potential to be industrially used only if they are associated with effective processes in which little exposure of the biopolymer to thermal degradation occurs. However, no mention is made to the properties of the materials obtained, especially those referring to the molecular weight of the product.
Other relevant fact regarding the industrial viability of this mode of PHA extraction is that, since it is a process of high energy consumption, we should bear in mind that the viability of the product is also intimately related to the availability of a low cost renewable source of energy.
Considering all the factors mentioned above, in general the properties of biodegradability and sustainability of the PHAs, although they can justify higher prices than those of the traditional polymers of the petrochemical industry, the possibility of the market to assimilate these prices is very limited (Braunegg G, Lefebvre G, Genser F K (1998) Polyhydoxyalkanoates, biopolyesters from renewable resources: Physiological and engineering aspects. J. Biotech. 65: 127-161).
Thus, industrial processes for producing PHAs should contemplate: strains of microorganisms that present high efficiency in the conversion of the raw material into polymer, with a simple and efficient production protocol; raw materials of low cost and high yield; a procedure of extraction and purification of the polymer which allows obtaining a product of high purity, preserving at maximum the original characteristics of the biopolymer, with high yield and efficiency and through processes that are not aggressive to the environment.
Besides these economical aspects, since it is an environmental friendly product, the whole process thereof should be compatible. Thus, the use of environmental harmful products in any production step should be avoided. Moreover, the source of energy used to run the process of production should come from a renewable source. It would not make sense to produce a plastic of low environmental impact if only non-renewable sources of energy are employed. A quite interesting approach to this problem is to have the entire productive chain of the bioplastic incorporated by the agro-industry, in particular by the sugar and alcohol industry (Nonato, R. V., Mantelatto, P. E., Rossell, C. E. V., “Integrated Production of Biodegradable Plastic (PHB), Sugar and Ethanol”, Appl. Microbiol. Biotechnol. 57:1-5, 2001).
One of the greatest worldwide successes in the production of alternative fuels is the sugar and alcohol industry in Brazil, which is responsible for about 25% the total amount of alcohol and sugar produced in the planet. While presenting an environmentally negative image in the beginning of the PROÁLCOOL Brazilian program, this type of industry is actually an example of sustainable technology. All energy required to run the production process is generated in loco, by burning the sugar cane bagasses in boilers to produce thermal and electric energy. Moreover, there is an excess of energy that can be used in other incorporated industrial processes.
A renewable and cheap energy allied with the availability of cheap raw materials, such as sugar and molasses and natural solvents obtained as by products of the alcoholic fermentation makes the sugar and alcohol industry the ideal cradle for the production of bioplastics.
Therefore, the present invention encompasses all the characteristics cited above which are necessary to make viable an industrial process for recovering polyhydroxyalkanoates (PHAs), preferably from humid bacterial biomass, using non-halogenated solvents which are not aggressive to the environment, generating a product of high purity and high molecular weight, by employing renewable raw materials and energy sources from the sugar and alcohol industry using sugarcane.